Astrocyte elevated gene-1对肾母细胞瘤生物学行为的影响及分子机制研究

发布时间：2018-01-26 12:56

本文关键词： 肾母细胞瘤 astrocyte elevated gene-1 慢病毒 RNA干扰　出处：《山东大学》2014年博士论文　论文类型：学位论文

【摘要】：研究背景和目的肾母细胞瘤,又称Wilms瘤,约占所有儿童肿瘤的比例为8%,是儿童比较常见的恶性肿瘤,同时也是儿童泌尿系统中最常见的恶性肿瘤。近些年来,虽然经过手术、化疗、放疗等综合治疗,肾母细胞瘤的生存率有所提高,但仍有部分患儿由于复发、转移以及对化疗药物的不敏感导致治疗效果不佳而死亡。因此,探讨肾母细胞瘤的发生发展机制,寻找肾母细胞瘤的分子生物学标记物,具有重要的意义。 Astrocyte elevated gene-1(AEG-1)是Su等在2002年首次克隆出来的新基因,最早发现于感染HIV病毒的PHFA (primary human fetal astrocytes)细胞。近年来对AEG-1的深入研究,发现AEG-1能激活多条信号转导通路,如PI3K/AKT, NF-κB、MEK/ERK、WNT/β-catenin等,与肿瘤的增殖、侵袭、转移、血管生成、抗凋亡、耐药等密切相关。尽管在多种体外培养的肿瘤细胞如胃癌、食管癌、肝细胞肝癌、恶性神经胶质瘤、神经母细胞瘤、子宫内膜癌、乳腺癌、黑色素瘤、肾细胞癌及前列腺癌等以及多种肿瘤组织标本中都发现AEG-1表达显著升高并对其机制进行了研究,但是,AEG-1与肾母细胞瘤中的相关性研究,国内外均未见报道。 RNA干扰(RNA interfering, RNAi)技术目前已广泛应用于基因功能、基因表达调控、基因治疗以及调控细胞行为等多个领域,成为重要的研究工具。由慢病毒介导的表达载体因其表达持久、安全性好等自身优势,在基因治疗的研究中得到了广泛的应用,并成为目前RNAi研究和转基因的最佳工具之一。基于以上研究背景,本课题研究AEG-1在肾母细胞瘤中的表达情况,及其与临床特征的关系及对病人预后的影响；构建靶向AEG-1特异性RNA干扰慢病毒表达载体,体外转染人高表达AEG-1的G401细胞株,沉默其AEG-1基因的表达,研究AEG-1基因被抑制后G401细胞增殖、运动、侵袭、周期、调亡、化疗敏感性等方面的影响并检测与之相关的蛋白水平,初步探讨其可能的分子机制,为临床寻找肾母细胞瘤基因治疗的新靶点提供借鉴。方法 1.AEG-1在肾母细胞瘤中的表达及其对预后的影响：选取山东省立医院肾母细胞瘤手术切除标本42例,应用免疫组织化学方法检测AEG-1的表达,并分析其与临床特征的关系及对病人预后的影响。 2.慢病毒介导的肾母细胞瘤细胞AEG-1基因沉默：采用实时荧光定量PCR(Real time PCR)及蛋白质印迹(western blot)技术检测人胚肾细胞株CCC-HEK-1和人肾Wilms瘤细胞株G401中AEG-1的表达情况。构建针对AEG-1基因的、带绿色荧光蛋白标记的、由U6启动子驱动的慢病毒RNAi载体,转染G401细胞。荧光显微镜观察绿色荧光蛋白的表达并确定转染效率,通过实时荧光定量PCR、western blot分别检测细胞转染后AEG-1mRNA和蛋白的水平变化情况,分析并确定RNA干扰的抑制效率,最终筛选出AEG-1沉默效率较高的干扰片段。 3.AEG-1基因沉默对肾母细胞瘤细胞生物学行为的影响及分子机制研究：将筛选出的最佳沉默效果的慢病毒载体转染G401细胞,沉默AEG-1的基因表达,通过MTT法检测AEG-1干扰后G401细胞增殖能力的变化；通过细胞划痕实验检测AEG-1沉默后G401细胞迁移能力变化,通过transwell小室侵袭实验检测AEG-1基因干扰后G401细胞的侵袭能力的变化,采用、vestern blot检测与肿瘤细胞侵袭转移相关的基质金属蛋白酶MMP2和MMP9蛋白表达的变化；应用流式细胞技术检测AEG-1沉默对G401细胞周期和凋亡的影响,采用western blot检测细胞周期相关蛋白Cyclin Dl、CDK2、p21Cip1、p27Kip1以及细胞凋亡相关蛋白Bcl-2、Bax、Caspase-3的表达变化；MTT法检测AEG-1沉默后G401细胞对化疗药物阿霉素和放线菌素D的敏感性变化情况,采用western blot检测耐药基因MDRl蛋白表达的变化。结果 1.AEG-1在肾母细胞瘤中的表达及其对预后的影响：在纳入研究的42例肾母细胞瘤中,肿瘤组织中AEG-1高表达21例(50%),而瘤周正常组织中检测不到AEG-1表达。AEG-1的表达情况在不同临床分期(P=0.019)、病理分型(P=0.048)、复发情况(P=0.015)的患儿之间有显著性差异,而在不同性别(P=0.751)、年龄(P=0.354)的患儿之间无显著性差异。AEG-1高表达组的5年DFS和OS均显著低于低表达组(P=0.006和P=0.007)。 2.慢病毒介导的肾母细胞瘤细胞AEG-1基因沉默：real time PCR和westernblot检测证实G401细胞中(?)EG-1mRNA和蛋白水平显著高于CCC-HEK-1细胞。以AEG-1mRNA基因序列为靶目标进行干扰片段设计,成功构建了2对针对AEG-1基因不同位点的特异性RNA干扰序列,经化学修饰后与pGCSIL-GFP慢病毒载体连接,通过转化,挑选阳性克隆进行测序,鉴定干扰片断的插入位点和碱基序列与设计相符；在293T细胞中包装慢病毒,获得高滴度慢病毒上清；利用慢病毒将AEG-1基因的RNA干扰片段转染G401细胞,并测定转染效率达80%以上,real time PCR和western blot检测证实与未转染组相比,干扰组(AEG-1RNAi-LV1和(?)EG-1RNAi-LV2)的AEG-1mRNA和蛋白水平显著降低,其中又以AEG-1RNAi-LV1的下降更明显,说明在2个干扰靶点中,(?)EG-1RNAi-LV1的干扰效果做好,后续实验选用该组。 3.AEG-1基因对肾母细胞瘤的生长、迁移、细胞周期及耐药性具有明显的调节作用：细胞划痕实验显示与未转染组相比,AEG-1干扰后细胞增殖能力不断下降,至第5天(120h)有显著性差异(P0.0001)；在各个时间点的划痕区域增宽,且在观察48h时有显著性差异(P=0.0413),该结果表明下调AEG-1抑制了细胞的侵袭转移,且有显著性差异(P=0.0238)；细胞侵袭转移能力的下降与MMP2和MMP9蛋白表达水平密切相关,MMP2与MMP9的表达明显下调,差异显著(P=0.0395,P=0.0213)；AEG-1具有调节细胞周期的能力,结果显示S期细胞比例明显减少,而G0/G1期细胞比例明显增多(P0.001),调节细胞周期的蛋白p21Cipl和p27Kip1表达显著增加(P0.001)；AEG-1的下调亦能诱导细胞的凋亡,流式细胞仪的检测结果表明细胞调亡比例增多(P=0.0024),而调控细胞凋亡的核心蛋白Bcl-2蛋白表达水平显著降低(P=0.024),Bax和Caspase-3蛋白显著增加(P0.001)；AEG-1也能影响肿瘤细胞对化疗药物耐药性的变化,结果表明AEG-1的低表达对阿霉素和放线菌素D的半数抑制浓度IC50均显著降低(P0.001),MDR1蛋白表达水平显著降低(P=0.0018)。结论 1.AEG-1的表达情况在不同临床分期、病理分型、复发情况的患儿之间有显著性差异,AEG-1高表达提示预后不良。 2.G401细胞的(?)EG-1mRNA和蛋白表达水平均显著高于对照组细胞。成功构建2种慢病毒系统介导AEG-1RNAi载体,可稳定表达于靶细胞G401,能下调其目的基因AEG-1的(?)RNA和蛋白表达,尤其以AEG-1-RNAi-LV1沉默效果更好。 3.沉默AEG-1基因后,G401细胞的增殖能力下降；通过降低MMP2和MMP9蛋白表达水平,使G401细胞的迁移能力和侵袭能力下降；通过增加p21Cip1和p27kip1蛋白表达水平,使G401细胞阻滞于G0/G1期；通过降低Bcl-2蛋白表达水平、增加Bax和Caspase-3蛋白表达水平,使G401细胞凋亡增多；通过降低MDRl/P-gp蛋白表达水平,提高了G401细胞对阿霉素和放线菌素D的敏感性。
[Abstract]:Background and purpose of the study Wilms tumor , also known as Wilms tumor , occupies about 8 % of all children ' s tumors . It is the most common malignant tumor in children . Astrocyte elevated gene - 1 ( AEG - 1 ) was the first new gene cloned from Su et al . in 2002 . It was found that AEG - 1 can activate multiple signal transduction pathways , such as the proliferation , invasion , metastasis , angiogenesis , apoptosis and drug resistance . RNA interfering RNA interference ( RNAi ) technology has been widely used in many fields such as gene function , gene expression regulation , gene therapy and regulation of cell behavior . On the basis of the above research background , this topic studies the expression of AEG - 1 and its relationship with clinical features and its influence on the prognosis of patients . The expression of AEG - 1 - specific RNA interference lentivirus expression vector was constructed . The expression of AEG - 1 gene in vitro was constructed . The expression of AEG - 1 gene was studied . The expression of AEG - 1 gene was studied . The expression of AEG - 1 gene was studied . The expression of AEG - 1 gene was studied . method 1 . The expression of AEG - 1 in nephrostomas and its effect on prognosis were studied : 42 cases were resected specimens from Shandong Provincial Hospital , the expression of AEG - 1 was detected by immunohistochemical method , and its relationship with clinical features was analyzed and the effect of AEG - 1 on prognosis was analyzed . 2 . The expression of AEG - 1 gene in human embryonic kidney cell line CCC - HEK - 1 and human kidney Wilms tumor cell line G401 was detected by real - time fluorescence quantitative PCR ( Real time PCR ) and Western blot . 3 . The effect of AEG - 1 gene silencing on the biological behavior of G401 cells was studied by MTT assay . The changes of cell proliferation ability after AEG - 1 gene silencing were detected by MTT assay . The changes of expression of cyclin Dl , CDK2 , p21 Cip1 , p27Kip1 and apoptosis related protein Bcl - 2 , Bax , Caspase - 3 were detected by MTT assay . Results 1 . The expression of AEG - 1 and AEG - 1 in the tumor tissues were significantly lower than those in the low expression group ( P = 0.019 ) , pathological type ( P = 0.048 ) and recurrence ( P = 0.015 ) . The expression of AEG - 1 was significantly lower in the patients with different clinical stages ( P = 0.019 ) , age ( P = 0.048 ) and recurrence ( P = 0.015 ) . 2 . The expression of AEG - 1 mRNA and protein in G401 cells was significantly higher than that of CCC - HEK - 1 cells . The interference effect of EG - 1RNAi - LV1 is well done , and the subsequent experiment selects the group . 3 . The growth , migration , cell cycle and drug resistance of the AEG - 1 gene were significantly lower than those in the untransfected group ( P = 0.02395 , P = 0.0213 ) . Conclusion 1 . The expression of AEG - 1 was significantly different among children with different clinical stages , pathological types and recurrence , and AEG - 1 high expression suggested poor prognosis . 2 . The expression level of ( ? ) EG - 1mRNA and protein in G401 cells was significantly higher than that in the control group . Two lentivirus systems were successfully constructed to mediate the expression of AEG - 1 RNAi vector , which could downregulate the expression of RNA and protein of AEG - 1 gene . Especially , the silencing effect of AEG - 1 - RNAi - LV1 was better . 3 . After silencing the AEG - 1 gene , the proliferation ability of G401 cells decreased ; by decreasing the expression level of MMP2 and MMP 9 , G401 cells were blocked in G0 / G1 phase ; by decreasing the expression level of p21 Cip1 and p27kip1 protein , the expression level of Bax and Caspase - 3 protein was increased , and the expression level of Bax and Caspase - 3 protein was increased , and the sensitivity of G401 cells to adriamycin and actinomycin D was improved by lowering the expression level of MDRI / P - gp protein .

【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R737.11

【共引文献】